The epigenetic modification of DNA is essential for normal development and differentiation in higher eukaryotes (reviewed in Robertson, Nat. Rev. Genet. 6(8): 597-610 (2005)). Mammalian DNA is modified at CpG residues through the addition of a methyl group to the cytidine nucleotide. Clusters of CpG dinucleotides are found in the promoter and 5′ regions of genes, and are referred to as CpG islands. Approximately half of mapped human genes have a CpG island upstream or within the 5′ end of the transcript. Methylation of gene-associated CpG islands is correlated with reduced expression or complete silencing of the corresponding gene (reviewed in Baylin, Nat. Clin. Pract. Oncol. Suppl. 1: S4-11 (2005)).
The ability to detect methylation patterns in DNA is desirable in many applications, such as in the context of diagnosis of diseases, such as cancer. Over the last 20 years, analysis of methylation patterns derived from tumor tissues has identified aberrant methylation (hypomethylation and/or hypermethylation) of CpG islands as a common event in most cancers (Lumd et al., Genes Dev. 18(19): 2315-2335 (2004); Baylin, Nat. Clin. Pract. Oncol. Suppl. 1: S4-11 (2005); and Laird, Hum. Mol. Genet. Spec. No. 1: R65-76 (2005)). This aberrant methylation represents a novel DNA modification, which is not normally present in normal tissue and may not be present in the organism. As such, the detection of aberrant methylation in tissue samples or in DNA recovered from bodily fluids is an indication of the presence of cancer. Methylation offers a rich target for diagnostics, given the large number of sequences that are affected. In fact, a CpG island does not need to be associated with the promoter or the 5′ end of a transcript to be useful in cancer diagnosis. The only requirement is for a sequence containing one or more CpG dinucleotides to exhibit a change in methylation during the development of a disease. Since both hypo- and hyper-methylation have been observed in cancer, tests can be developed to detect either/both methylation states in the diagnosis of cancer as well as other disease states.
There are a number of obstacles that need to be overcome in order to utilize successfully the detection of DNA methylation in diagnostic applications. First, the amount of DNA recovered from bodily fluids, such as plasma or urine, is usually only a few nanograms per milliliter (Jahr et al., Cancer Res. 61(4): 1659-1665 (2001); and Anker et al., Cancer Metastasis Rev. 18(1): 65-73 (1999)), which is insufficient for the analysis of a multitude of markers. Some cancer patients can exhibit higher levels of DNA in their bodily fluids, while others have amounts comparable to those isolated from normal individuals. While the amount of DNA can be increased by extracting a larger sample, there is a limit as to the volume that can be obtained and analyzed in a clinical setting.
Second, circulating DNA can be derived from both normal and diseased tissues. When nucleic acids are isolated from tissue samples and cell lines, the purified DNA is representative of the sample from which it was isolated. In normal tissues, genomic markers are equally represented in the purified DNA. The same may not be true for circulating DNA. The amount of circulating DNA derived from cancer cells can be very small and can represent as little as 3% of the total (Jahr, supra). In addition, the minimal amount of circulating DNA may not contain an equal representation of all abnormally methylated CpG islands present in the tumor.
Third, circulating cancer DNA can be derived from apoptotic and necrotic cells and, therefore, can be degraded and/or severely fragmented. The DNA fragments can be as short as a single or few nucleosomes (about 150 bp or short multiples of 150 bp). Most CpG islands are longer than 250 bp and many are longer than 1 kb. Fragmentation of DNA into nucleosomes causes the majority of CpG islands to be cleaved into 2 or more segments.
Fourth, the DNA can be further degraded by the methods used to reveal the methylation pattern. The most common method involves the controlled treatment of DNA with sodium bisulfite (Frommer et al., PNAS 89: 1827-1831 (1992)), which preferentially deaminates unmethylated cytidines, resulting in a detectable sequence change. Bisulfite treatment is harsh and results in severe degradation and loss of DNA (Grunau et al., Nucleic Acids Res. 29(13): E65-5 (2001)).
Fifth, cancer detection requires the use of multiple markers (Yegnasubramanian et al., Cancer Res. 64(6): 1975-86 (2004)). Even more markers will be needed in cancer diagnostic assays from circulating DNA to achieve the sensitivity and specificity needed for clinical applications. Methylation analysis is further complicated by the heterogeneity of DNA methylation patterns in cancers which may differ between tumors derived from different tissues (Esteller, Cancer Res. 61(8): 3225-9 (2001)), and between tumors derived from the same tissue (Zhao, Cancer 104(1): 44-52 (2005)). Therefore, methylation-based cancer diagnosis requires the analysis of multiple CpG islands and multiple CG residues across the length of an island. Such analysis requires more DNA than is usually isolated from bodily fluids.
Given all of these limitations, the detection of CpG methylation from circulating DNA requires assays that are designed to improve the sensitivity of detection despite the fragmentation of the templates. There is a need for a method that preserves the methylation pattern of the DNA, while, at the same time, increasing the amount of the DNA, so that methylation patterns can be easily detected and used to diagnose diseases. In this regard, methods to amplify DNA are well-known in the art and generally fall under two broad categories: the polymerase chain reaction (PCR) and isothermal amplification (see various patents and published patent applications by Lizardi, such as U.S. Pat. Nos. 5,854,033; 6,124,120; 6,143,495; 6,210,884; 6,642,034; 6,280,949; 6,632,609; and 6,642,034; and U.S. Pat. App. Pub. Nos. 2003/0032024; 2003/0143536; 2003/0235849; 2004/0063144; and 2004/0265897, each of which is specifically incorporated herein by reference in its entirety).
PCR requires the use of thermostable polymerases and primers flanking regions of interest and repeated cycling between temperatures that allow for denaturing of the DNA, annealing of primers to complementary DNA, and replicating of targeted sequences. When the primers used are random or partially random, they anneal to homologous sites randomly across the entire genome, leading to whole genome amplification.
Isothermal amplification, on the other hand, relies on the ability of mesophilic and thermophilic polymerases to displace existing DNA strands during DNA replication. When multiple primers, which flank regions of interest, are added to the amplification mix, multiple strand displacements lead to the generation of numerous overlapping fragments corresponding to the regions of interest. When the DNA is circular, strand displacement results in amplification by rolling circles. When random primers are used, multiple strand displacements lead to amplification of the whole genome. In addition, primers, which contain unique and random sequences, can be used to introduce unique “tags” across the genome. The advantage of isothermal amplification is the ability to perform the entire amplification process under constant temperature.
Methods, which combine PCR and isothermal amplification, also have been described (U.S. Pat. Nos. 6,777,187; and 6,828,098; and U.S. Pat. App. Nos. 2004/0209298; 2005/0032104; and 2006/0068394, each of which is specifically incorporated herein by reference in its entirety). Makarov et al. (U.S. Pat. App. No. 2005/0202490, which is specifically incorporated herein by reference in its entirety) describes the use of such methods in combination with methylation-sensitive restriction enzymes to study the methylation pattern of DNA.
All DNA amplification methods, however, are disadvantageous in that they fail to copy the methylation pattern of the DNA. Thus, in and of themselves, they are useless for the detection of methylation in the diagnosis of disease.
Lopez et al. (U.S. Pat. No. 6,514,698 and Int'l Pat. App. Pub. No. WO 99/10540) discloses using DNA methyltransferases to introduce methylation patterns (i.e., de novo methylation) into amplified DNA to detect variations, mutations or polymorphisms in DNA in an effort to genotype the DNA. Lopez et al. discloses that the use of DNA methyltransferases as proposed overcomes many of the disadvantages of the use of restriction endonucleases to genotype DNA. Lopez et al. does not teach, let alone even suggest, that DNA methyltransferases could be used to copy existing methylation patterns, while amplifying DNA, in an effort to assess abnormal methylation in the diagnosis of disease.
Berlin et al. (Int'l Pat. App. Nos. PCT/EP03/03104 and PCT/EP03/03105, Int'l Pub. Nos. WO 03/080862 and WO 03/080863, which are specifically incorporated herein by reference in their entireties) discloses that mass spectrometry can be used to analyze the methylation patterns of preselected regions (or targets) of amplified genomic nucleic acids, which have been isolated from a biological sample, and sequentially (i) copied and (ii) methylated repeatedly until the desired amount of nucleic acid is obtained. Berlin et al. discloses that ligase chain reaction, polymerase chain reaction, polymerase reaction, or rolling circle replication can be used to copy the preselected regions (or targets). However, while the methylation patterns of the preselected regions (or targets) of the genomic nucleic acids are maintained with sequential copying and methylation reactions, Berlin et al. does not enable maintenance methylation under isothermal amplification conditions when rolling circle replication is used or when polymerases with strand-displacement capabilities are used. The only conditions taught (pg. 29, lines 20-24) by Berlin et al. for methylation reaction with DNA methyltransferase 1 (DNMT-1) do not enable the polymerization reaction with a strand-displacing polymerase. Furthermore, Berlin et al. fails to address the de novo methylation that is introduced into the sample when DNMT-1 is used to copy the methylation pattern. The de novo activity of DNMT-1 introduces methylation on templates, which are not methylated in the original sample. This activity mimics the presence of a small amount of methylated templates as would be expected when circulating DNA from patients with early onset of disease is analyzed. The methodology of Berlin et al. will not enable the early detection of disease, such as cancer, because the amount of DNA that is derived from circulating DNA is minute, often representing less than 5% of the total DNA.
Fodor et al. (U.S. Pat. App. Pub. No. US 2005/0196792, which is specifically incorporated herein by reference in its entirety) discloses the use of a strand-displacing polymerase and a DNA methyltransferase to copy the methylation pattern from the parent strand into the daughter strand before the daughter strand is displaced during replication. The only conditions taught (para. [0076]) by Fodor et al. for methylation reaction with the DNMT-1 do not enable the polymerization reaction with a strand-displacing polymerase. Furthermore, like Berlin et al., Fodor et al. fail to address the de novo methylation that is introduced into the sample when DNMT-1 is used to copy the methylation pattern. Consequently, Fodor et al. also will not enable the early detection of disease, such as cancer. Furthermore, while Fodor et al. may claim variant forms of human and mouse DNMT-1s, Fodor et al. does not teach, let alone suggest, how to obtain such variant forms.
In view of the above, there remains a need for a method that enables copying of the methylation patterns of DNA molecules during isothermal amplification using a DNA methylation-maintenance enzyme and a DNA polymerase with strand displacement activity under conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase with strand displacement activity. Preferably, the method provides a level of sensitivity to detect methylation patterns in DNA available only in very limited quantities. The present invention seeks to provide such a method. This and other objects and advantages, as well as additional inventive features, will become apparent from the detailed description provided herein.